A variety of endonucleases, designated “restriction enzymes” or “restriction endonucleases,” are used in the art to cleave double-stranded DNA. These enzymes bind to specific sequences of DNA (the “recognition site”) and cleave the DNA either at the recognition site or at a site that is some distance away from the recognition site.
Although restriction enzymes are an important and widely-used tool in molecular biology applications, the use of restriction enzymes has certain limitations resulting from the functional properties of the restriction enzymes. First, the locations at which restriction enzymes can cleave a given double-stranded DNA molecule are limited to the specific nucleotide sequences on the DNA molecule that correspond to the recognition sites of available restriction enzymes. A given restriction enzyme will cleave DNA only at or a certain distance from a specific DNA sequence corresponding to the restriction enzyme recognition site. Although different restriction enzymes may have different recognition sites, there are a limited number of available restriction enzymes, and thus a limited number of recognition sites at which double-stranded DNA can be cleaved. If cleavage is desired at a certain pre-determined location on the DNA molecule that does not contain a known restriction enzyme recognition site, such a site must be engineered into the DNA molecule, which can be a difficult and time-consuming task.
Second, restriction enzymes often cleave double-stranded DNA at more than one location, even if cleavage is desired at only a single location. Because restriction enzyme recognition sites generally have relatively short nucleotide sequences (e.g. 4-9 nucleotides), a double-stranded DNA molecule may frequently contain a given recognition site at multiple locations. In such a case, the use of restriction enzymes to cleave a double-stranded DNA molecule at a target location may result in cleavage at both the target location and at additional recognition sites where cleavage is not desired.
Zinc finger endonucleases (ZFN) have been used in gene therapy applications to introduce double strand breaks at a specific chromosomal locus and to induce homology-directed repair with an exogenously added donor DNA sequence (Scott, 2005). However, the use of this technology is limited by the need to generate a new ZFN for each specific knockdown target, which is a difficult and expensive task.
Thus, there is a need in the art for a method of cleaving a double-stranded DNA molecule at a pre-determined location in a sequence-directed manner, without requiring either the generation of a novel ZFN or the engineering of a restriction enzyme recognition site at the pre-determined location.